This proposal focuses on several DNA metabolic pathways in eukaryotes, with an emphasis on DNA replication and DNA damage response mechanisms. DNA polymerases ? (Pol ?) and ? (Pol ?) carry out the elongation and maturation of Okazaki fragments on the lagging strand of the replication fork, and translesion synthesis (TLS), respectively. Our recent discovery that their catalytic subunits contain an essential iron-sulfur cluster makes new functional studies of these enzymes both timely and important. Thus, we will determine whether the redox state of the iron-sulfur cluster affects the biochemical parameters of these enzymes, either in catalysis or complex interactions. We will test the hypothesis that regulated strand displacement synthesis by Pol ? is a critical aspect of its function during Okazaki fragment maturation. During this process, which occurs millions of times during each mammalian cell division, strand displacement synthesis by Pol ? generates 5'-flaps that are cut by the flap endonuclease FEN1. The kinetic mechanism of this machinery will be determined, and their physiological relevance will be queried through genetic analysis of informative mutants. Our proposed studies of mutagenesis focus on Rev1 and Pol ?, which through interactions with the replication clamp PCNA form a productive mutasome. The Rev1 protein serves as a scaffold onto which the TLS machinery is organized. We will test the hypothesis that posttranslational modifications act as on/off switches for mutagenesis by mediating functional interactions between Pol ? and Rev1 with PCNA. The primary focus of our checkpoint studies will be on the sensor protein kinase Mec1, the ortholog of human ATR, that initiates the DNA damage and replication checkpoints. The initiating step in these pathways is the activation of Mec1/ATR kinase activity through interaction with cell-cycle specific activators on single-stranded DNA substrates coated with the single-stranded binding protein RPA. Key features of these activators is that the activating regions are structurally disordered and contain small essential aromatic amino acid motifs. Biochemical studies are aimed at understanding Mec1 kinase activation by these proteins and complexes. In keeping with the MIRA principle, we also look forward to pursuing other fascinating questions in DNA metabolism that may, and undoubtedly will arise during our investigations.